CAP37 activation of PKC promotes human corneal epithelial cell chemotaxis.

PURPOSE: The objective of this study was to elucidate the signaling pathway through which cationic antimicrobial protein of 37 kDa (CAP37) mediates human corneal epithelial cell (HCEC) chemotaxis. METHODS: Immortalized HCECs were treated with pertussis toxin (10 and 1000 ng/mL), protein kinase C (PKC) inhibitors (calphostin c, 50 nM and Ro-31-8220, 100 nM), phorbol esters (phorbol 12,13-dibutyrate, 200 nM and phorbol 12-myristate 13-acetate, 1 µM) known to deplete PKC isoforms, and siRNAs (400 nM) before a modified Boyden chamber assay was used to determine the effect of these inhibitors and siRNAs on CAP37-directed HCEC migration. PKCδ protein levels, PKCδ-Thr(505) phosphorylation, and PKCδ kinase activity was assessed in CAP37-treated HCECs using immunohistochemistry, Western blotting, and a kinase activity assay, respectively. RESULTS: Chemotaxis studies revealed that treatment with pertussis toxin, PKC inhibitors, phorbol esters, and siRNAs significantly inhibited CAP37-mediated chemotaxis compared with untreated controls. CAP37 treatment increased PKCδ protein levels and led to PKCδ phosphorylation on residue Thr(505). Direct activation of PKCδ by CAP37 was demonstrated using a kinase activity assay. CONCLUSIONS: These findings lead us to conclude that CAP37 is an important regulator of corneal epithelial cell migration and mediates its effects through PKCδ.

Modulation of corneal epithelial cell functions by the neutrophil-derived inflammatory mediator CAP37.

PURPOSE: To investigate the effect of CAP37, an inflammatory mediator in neutrophils, on three important events in corneal wound healing: proliferation, migration, and adhesion. METHODS: Immortalized human corneal epithelial cells (HCEC) were treated with CAP37, and its effects on migration and proliferation were measured using the modified Boyden chemotaxis chamber and the proliferation assays (CyQUANT; Molecular Probes, Eugene, OR), respectively. Effects on adhesion were determined by measuring upregulation of adhesion molecules belonging to the selectin, integrin, and immunoglobulin superfamily using RT-PCR and flow cytometry. RESULTS: CAP37 promoted proliferation of HCEC in a time- and dose-dependent fashion. CAP37 was maximally chemotactic for HCEC over a range of 1.3 x 10(-8) to 5.2 x 10(-8) M. CAP37 upregulated intercellular adhesion molecule (ICAM)-1, platelet endothelial cell adhesion molecule (PECAM)-1, and integrin molecules alpha3 (CD49c) and beta1 (CD29). Data on migration and ICAM-1 and PECAM-1 upregulation were corroborated using primary human corneal epithelial cells. CONCLUSIONS: CAP37 modulated corneal epithelial cell proliferation and migration and upregulated adhesion molecules involved in leukocyte-epithelial and epithelial-extracellular matrix interactions.

Functional modulation of smooth muscle cells by the inflammatory mediator CAP37.

CAP37, a neutrophil-derived protein, originally identified for its antimicrobial activity is now known to have strong immunoregulatory effects on host cells. Recently, we described its expression and localization within the vascular endothelium associated with atherosclerotic plaques. Since CAP37 is a potent activator of endothelial cells and monocytes, two of the key cellular components of the atherosclerotic plaque, this study was undertaken to determine whether CAP37 had functional effects on smooth muscle cells another important cellular participant in atherosclerosis. Sections from atherosclerotic lesions were stained for the presence of CAP37 and smooth muscle cell alpha actin. The effect of CAP37 on aorta smooth muscle cell migration and proliferation was investigated and the upregulation of adhesion molecules was determined. Immunocytochemistry indicated that CAP37 was present in a subset of smooth muscle cells within atherosclerotic lesions, but was absent in normal vessels. Flow cytometry using double labeling for the proliferation marker Ki-67 and CAP37 demonstrates that CAP37 is mainly expressed in proliferating smooth muscle cells. We show that CAP37 supports migration and proliferation of smooth muscle cells in vitro. Furthermore, CAP37-treated smooth muscle cells expressed higher levels of the cell adhesion molecule ICAM-1 when compared with untreated cells. We suggest that due to its localization to atherosclerotic plaques and its ability to modulate smooth muscle cells, CAP37 may play a role in the progression of this disease.

Structural analysis of IgG2A monoclonal antibodies in relation to complement deposition and renal immune complex deposition.

This study explores the structural features of murine monoclonal IgG2a anti-dinitrophenyl (DNP) antibodies that were previously shown to form immune complexes (IC) differing in their capacity to bind complement, their clearance from the circulation and their deposition in the kidney. Interestingly, the sequence of one of these antibodies has a missing stretch of 14 amino acids within FR3. Molecular modeling suggests that this sequence deletion corresponds to the loss of beta-pleated sheet structure for two beta-strands (designated 4-3 and 4-4) on the external surface of the V(H) domain. Despite this sequence and conformational abnormality, the antibody retains affinity for DNP comparable to other IgG2a antibodies. Data presented here identify monoclonal IgG2a antibodies that form IC with varying propensity for both complement binding and renal deposition and yet have similar V(H) domain sequences. In fact, in the case of two IgG2a antibodies that form IC with very different renal tropisms and complement binding capacity, sequence variation within V(H) was observed only at three clustered residues within FR2, a single residue within FR3 and nine clustered residues spanning CDR3 and FR4. Sequence and modeling analysis also yielded the paradoxical finding that an antibody forming IC with a relatively high capacity to serve as a target for complement binding displays a relatively low number of solvent exposed acceptor residues for C4b and C3b. These data underscore the complex relationship between V domain structure, complement activation and renal deposition of model IC.

CAP37, a neutrophil-derived inflammatory mediator, augments leukocyte adhesion to endothelial monolayers.

Cationic antimicrobial protein of molecular weight 37 kDa (CAP37) is a multifunctional inflammatory mediator that was originally isolated from human neutrophils and described to possess bactericidal and monocyte-activating functions. More recently its expression in endothelial and epithelial cells in response to inflammatory mediators and its ability to activate endothelial cells and alter permeability has been demonstrated. We hypothesize that CAP37 facilitates the process of transendothelial migration not only because of its potential to act as a chemoattractant but also through its ability to promote leukocyte adhesion to the endothelium by modulating adhesion molecule expression on the endothelium. Here we describe its ability to mediate neutrophil and monocyte adherence to endothelial monolayers in vitro. Using reverse transcriptase-polymerase chain reaction and flow cytometry, we demonstrate its ability to upregulate the adhesion molecules, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin in human umbilical vein and lung microvessel endothelial cells. The identity and kinetics of upregulation of the specific adhesion molecule was dependent on the endothelial cell type, suggesting that adhesion molecules on endothelial cells from different vascular beds are differentially regulated by CAP37. The cell-specific kinetics of adhesion molecule upregulation by CAP37 may influence selective leukocyte migration in certain inflammatory situations.

CAP37, a novel inflammatory mediator: its expression in endothelial cells and localization to atherosclerotic lesions.

Cationic antimicrobial protein of 37 kd (CAP37), originally isolated from human neutrophils, is an important multifunctional inflammatory mediator. Here we describe its localization within the vascular endothelium associated with atherosclerotic plaques. Evidence from in vitro immunocytochemical, Northern blot, and reverse transcriptase-polymerase chain reaction analysis indicates that CAP37 is induced in endothelial cells in response to inflammatory mediators. Endothelial-derived CAP37 shows sequence identity with an extensive region of neutrophil-derived CAP37. This is the first demonstration of endogenous endothelial CAP37, confirmed by sequence analysis. We suggest that, because of its induction and location in the endothelium and its known monocyte- and endothelial-activating capabilities, CAP37 has potential to modulate monocyte/endothelial dynamics at the vessel wall in inflammation.